Efficient and precise replication of DNA is pivotal to the maintenance, transmission and expression of genetic information. High-fidelity DNA polymerases are the key enzymes responsible for maintaining genome integrity. To avoid the negative consequences of mutations (hereditary and sporadic diseases) high-fidelity DNA polymerases perform an astonishing feat of molecular recognition, selecting the correct nucleotide triphosphate (dNTP) molecule from a pool of very similar substrates and catalysing its incorporation as specified by the template base. DNA synthesis by exonucleolytic proof-reading deficient DNA polymerases occurs with error rates ranging from 10−3 to >10−6 per base-pair (Kunkel and Bebenek, 2000; Tippen et al., 2004). Although in nature high polymerase fidelity is vital for accurate DNA replication it has serious drawbacks for many biotechnological applications. Specifically, it restricts the use of unnatural or modified nucleotide bases and the applications they enable.
Fluorescence-based technologies have superseded radioisotopic detection as the preferred choice for labelling and detecting biomolecules. The incorporation of fluorescent labelled nucleotides into nucleic acids is central to such techniques as DNA sequencing, gene expression microarray analyses (Schena et al., 1995) tissue microarrays (TMA; Kononen et al., 1998), comparative genome hybridisations (CGH; Kallioniemi et al., 1992) and Fluorescent In-Situ Hybridisation (FISH; McNeil et al., 1991). Nucleic acid fluorescent labelling methodologies have been developed based on either enzymatic (direct incorporation of the fluorescent dye) or chemical modification (indirect incorporation of the fluorescent dye).
Direct incorporation of dye labelled nucleotides employs polymerase enzymes and is limited by the fact that polymerase enzymes have evolved to preserve a high selectivity for their correct nucleotide substrate. As such, most naturally occurring or commercially available polymerase enzymes discriminate against nucleotides bearing bulky side groups such as fluorescent moieties or incorporate them to a low level exhibiting significant sequence bias (Zhu and Waggoner, 1997; Zhu et al., 1994). Most enzymatic fluorescent labelling protocols, therefore, use the dye-labelled nucleotide, spiked at low percentage into a standard reaction mix (Reid et al., 1992). Even under these conditions, however, the polymerase enzyme still favours the natural nucleotide over the modified nucleotide (Zhu et al., 1994).
To overcome the low fluorophore densities achieved with direct labelling methods indirect labelling technologies have been developed whereby a less bulky amine-modified nucleotide is directly incorporated into the nucleic acid and the fluorescent label is chemically coupled to the nucleotide via the reactive amine group post nucleic acid synthesis (Cox and Singer, 2004). Although higher fluorescent nucleic acid labelling densities can be attained by indirect labelling methods complete substitution of every reactive nucleotide has not been achieved.
Nucleic acid probes with a higher density of labels (up to 100% substitution) are desirable as they would be expected to increase detection sensitivity. Furthermore, 100% substitution of every base with its fluorescently modified counterpart is a prerequisite of many single molecule sequencing techniques (Shendure et al., 2004). With current indirect DNA labelling methods unable to label 100% of the available positions research efforts have focussed on identifying naturally occurring or mutant DNA polymerase enzymes that are less stringent with regard to their substrate specificity.
Such efforts have met with modest success. Specifically, several members of the A (PolI-like; Brakmann and Nieckchen, 2001; Anderson et al., 2005; Yu et al., 1994; Tasara et al., 2003; Augustin et al., 2001; Ghadessy et al., 2004; Ramanathan et al., 2005) or B (PolII-like; Anderson et al., 2005; Augustin et al., 2001; Tasara et al., 2003; Földes-Papp et al., 2001; Glick et al., 2002; Jäger and Famulok, 2004; Obayashi et al., 2002; Ono et al., 1997) evolutionary families of DNA polymerase enzymes (Zhu and Ito, 1994) have been identified as able to incorporate fluorescent labelled nucleotides. In the case of enzymes harbouring polymerase as well as proofreading activity the yield of dye-labelled DNA was improved by using their exonuclease deficient mutants.
The ability of the majority of the polymerase enzymes to incorporate fluorescently labelled nucleotides was only investigated by primer extension analyses generating single stranded fluorescently labelled DNA. PCR incorporation of fluorescently labelled nucleotides allows simultaneous labelling and amplification of DNA. However, in contrast to primer extension reactions, following a few cycles of PCR amplification the fluorescent nucleotide is also present in the template strand. This has consequences for PCR amplification as the polymerase frequently pauses or aborts copying and the yield of labelled DNA decreases as the fluorescent nucleotide incorporation increases presumably due to steric crowding effects (Zhu and Waggoner, 1994). Consequently there is a need for polymerase enzymes able to efficiently incorporate fluorescent nucleotide analogues to high density by PCR and there remains a need in the art for polymerases, in particular DNA polymerases which are capable of incorporating a high density of detection label and/or capable of incorporating the detection label into large double stranded DNA fragments.